Involvement of DAAO Overexpression in Delayed Hippocampal Neuronal Death

Background: D-amino acid oxidase (DAAO) is a flavoenzyme that specifically catalyzes the deamination of many neutral and basic D-amino acids. This study aims to explore the pathological increment of hippocampal DAAO and its potential relationship with delayed hippocampal neuronal death. Methods: Ischemia–reperfusion was induced in mice through middle cerebral artery occlusion (MCAO). Neurological deficit scores and hippocampal neuronal death were assessed in MCAO mice. Immunofluorescent staining was applied to identify activated astrocytes and evaluate DAAO expression. TUNEL and Nissl staining were utilized to identify cell apoptosis of hippocampal neurons. Results: Hippocampal astrocytic DAAO was strikingly increased following ischemic stroke, with the greatest increase on day 5 after surgery, followed by the manifestation of neurobehavioral deficits. Astrocytic DAAO was found to be mainly expressed in the hippocampal CA2 region and linked with subsequent specific neural apoptosis. Thus, it is supposed that the activation of astrocytic DAAO in ischemic stroke might contribute to neuronal death. An intravenous, twice-daily administration of 4H-furo[3,2-b]pyrrole-5-carboxylic acid (SUN, 10 mg/kg) markedly relieved behavioral status and delayed hippocampal neuronal death by 38.0% and 41.5%, respectively, compared to the model group treated with saline. In transfected primary astrocytes, DAAO overexpression inhibits cell activity, induces cytotoxicity, and promotes hippocampal neuronal death at least partly by enhancing H2O2 levels with subsequent activation of TRP calcium channels in neurons. Conclusions: Our findings suggest that increased hippocampal DAAO is causally associated with the development of delayed neuronal death after MCAO onset via astrocyte–neuron interactions. Hence, targeting DAAO is a promising therapeutic strategy for the management of neurological disorders.

Introduction
D-Amino acids are important neurotransmitters that play key functions in the nervous system. The flavoprotein D-amino acid oxidase (DAAO) is the main metabolic enzyme of neutral and basic D-amino acids. The physiological roles identified for DAAO include regulation of the nervous system and hormone secretion, among others, as well as involvement in the development of different diseases resulting from alterations in DAAO activity. In the central nervous system, the pathophysiological functions of DAAO are implicated in hyperalgesia, morphine antinociceptive tolerance, and cognitive impairment, such as in the context of schizophrenia or other neurodegenerative diseases [1][2][3][4]. In postmortem autopsies, hippocampal DAAO levels are significantly higher in schizophrenia patients than in people without schizophrenia and are closely linked with disease course. Moreover, the expression of DAAO is more than 70% higher in long-term (>20 years) schizophrenia patients than people without schizophrenia [5]. Hence, DAAO inhibitors have been applied in schizophrenia therapy in a number of subsequent clinical studies [6,7]. In 2013, the first clinical report of DAAO inhibitor sodium benzoate use in the treatment of patients with chronic schizophrenia was published. Six weeks of treatment significantly improved patient cognition and alleviated other symptoms, and the drug was well tolerated (clinical trial registration number: NCT 00960219). A randomized, double-blinded, placebo-controlled clinical trial found that peripheral DAAO levels in dementia patients with psychobehavioral symptoms (BPSDs) are positively correlated with Alzheimer's Behavioral Scale scores. DAAO inhibitors could significantly improve cognitive function in patients with BPSDs (clinical trial registration number: NCT02103673) [8]. It has been demonstrated that DAAO expression is upregulated in a variety of neurodegenerative diseases, and DAAO-targeted therapies have resulted in improvements regarding clinical symptoms.
Our previous report was the first to disclose that DAAO expression steadily increases in activated astrocytes after ischemic stroke onset [9]. DAAO inhibitors or siRNA/DAAO could effectively alleviate brain injury by suppressing levels of the byproduct hydrogen peroxide, which implies that DAAO might be involved in the process of ischemic stroke. D-Ser is found at high physiological levels in the cerebrum and can be degraded by DAAO to increase levels of the byproduct hydrogen peroxide, which is one of the most stable reactive oxygen species (ROS), thus causing cytotoxicity to induce neuronal apoptosis as well as to reduce the expression of brain-derived neurotrophic factor (BDNF) [10,11]. In our previous study, we reported that cortical DAAO is upregulated following MCAO and is predominantly expressed in astrocytes. However, the cell specificity of hippocampal DAAO expression remains unknown. In addition, application of the DAAO inhibitor SUN reduces neurological deficits following MCAO, but the specific downstream mechanisms for this remain unclear. In this study, we focus on astrocytic DAAO expression in the hippocampus and whether it is related to delayed neuronal death after ischemic insult, which also needs clarification regarding astrocyte-neuron interactions. Furthermore, we subsequently investigate whether astrocytic DAAO in the hippocampus influences neurological deficits through its effect on neuronal survival. This study uses fluorescent probes to explore neurotoxicity and its underlying mechanism of hippocampal DAAO induction in response to acute neuropathological insult. Here, pcDNA3.1(+)-daao-transfected primary astrocytes are used to mimic the pathological overexpression of astrocytic DAAO in order to explore the causal relationship between DAAO and delayed neuronal death.

Animals
Specific-pathogen-free Swiss mice (male, >8 weeks, 20-22 g) were purchased from the Shanghai Experimental Animal Institute for Biological Sciences (Shanghai, China). The mice were reared in cages with free access to food and water following the animal care guidelines of the NIH (Bethesda, MD, USA). The animal houses used have an authorized biosafety license and were programmed to ensure a temperature-controlled environment with a normal 12 h light-dark cycle. The animal protocols were approved by the Animal Care and Welfare Committee of Shanghai Jiao Tong University (Shanghai, China) and were performed in accordance with the Guide for Care and Use of Laboratory Animals and the Animals in Research: Reporting In Vivo Experiments guidelines [12,13].

Mouse MCAO Model
As the most popular ischemic stroke model, transient MCAO was executed using a modified intraluminal occlusion technique with a monofilament as in our previous report [9]. In brief, mice were intraperitoneally injected with 1.5% pentobarbital sodium (Aladdin Biochemical Technology, Shanghai, China) before surgery. After the common carotid artery was exposed and ligated to stop bleeding, a 6-0 nylon monofilament coated with a silicone tip (Rayward Life Technology, Shenzhen, China) was inserted into the common carotid artery to a position about 0.8-0.9 cm deep starting from the bifurcation site for occlusion of the middle cerebral artery. The monofilament was fixed to block the middle cerebral artery for 90 min in an incubator at 32 ± 0.5 • C and withdrawn for subsequent reperfusion. The exclusion criteria were as follows: (1) unexpected death; (2) no infarct injury or only a small infarct size; and (3) intracranial hemorrhage [14]. Following implementation of these criteria, around 60% to 80% mice remained. All ischemic mice were randomly divided into each group (n = 4-6), as indicated in the legend figures, according to simple randomization based on body weight (range from 20 to 22 g). The mice in the sham group were subjected to the same surgical process without inserting the intraluminal monofilament for 5 days following pseudo-surgery. Approximately 20% of the ischemia-reperfusion-induced mice were excluded due to death on days 4 and 5 after surgery. The following steps were undertaken to study the involvement of hippocampal DAAO in ischemic strokes: (1) Many indicators were measured to assess the relationships between DAAO expression and pathological changes in MCAO mice on days 0, 1, 3, and 5 after ischemia-reperfusion. These subgroups were defined according to the timepoint after surgery. Normal mice were defined as MCAO mice on day 0. (2) The DAAO inhibitor SUN was administrated to further evaluate efficacy. The mice were divided into a sham group, an MCAO control group (MCAO mice treated with saline only), and an MCAO treatment group (MCAO mice administered 10 mg/kg DAAO inhibitor SUN in intravenous, twicedaily injections). All of the described assays were conducted on samples from this study.

Behavioral Deficit Score
Neurobehavioral status was evaluated after ischemic stroke using the Zea Longa neurological grading scale (0-5) [15]. Grades were scored by the deficit degree of hemiparesis in contralateral limbs: 0 = no observable deficit; 1 = flexural forepaw on ipsilateral side; 2 = walking in circle; 3 = falling when walking; and 4 = unable to walk with depression status.
To examine time courses of immunolabeled DAAO in the hippocampal CA2 area, cerebral slices were collected from MCAO mice on days 0, 1, 3, and 5 after ischemia-reperfusion. To quantify the immunofluorescent intensity of cerebral slices in the hippocampal CA2 area, fields of merged images were randomly selected under a confocal microscope with 20× or 40× magnification, and ImageJ software (National Institutes of Health, Bethesda, MD, USA) was applied blindly by an investigator to measure the colocalized pixels of immunostaining areas accordingly. The quantified area fractions are represented as the ratio of the colocalized area to the total scanned area.

Western Blot
Samples in these tests were extracted from the hippocampal tissue of MCAO mice on days 0, 1, 3, and 5 after ischemia-reperfusion or from pcDNA3.1-transfected primary astrocytes. Western blotting was conducted to detect DAAO expression of hippocampal sections or primary astrocytes, as previously described [9]. Samples were extracted using RIPA buffer containing a cocktail of phenylmethylsulfonyl fluoride protease inhibitor and phosphatase inhibitor (50×, Beyotime Institute of Biotechnology, Nantong, China), prepared for electrophoresis by mixing with loading buffer (5×, Beyotime Institute of Biotechnology, Nantong, China), and denatured in a metal bath at 105 • C for 12 min. The protein concentration in the homogenate was determined with a bicinchoninic acid protein method (Beyotime Institute of Biotechnology, Nantong, China). The amount of total protein in the loading sample was 30-50 µg. DAAO antibody (1:3000, tailored polyclonal antibody from Novus Biologicals, Centennial, CO, USA) was used to label the target protein, and β-actin antibody (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA) was applied as a reference protein. After fluorescence-labeling with secondary antibodies (Dylight 680conjugated anti-mouse IgG (1:10,000) and Dylight 800-conjugated anti-rabbit IgG (1:10,000); Cell Signaling Technology, Danvers, MA, USA), protein bands were visualized using an Odyssey Infrared Imaging system (Li-Cor Biosciences, Lincoln, NE, USA) and quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

DAAO Activity Assay
The hippocampal DAAO activity of MCAO mice was further observed through KYNA and hydrogen peroxide, both of which are byproducts of the oxidative deamination of Dkynurenine catalyzed by DAAO. In accordance with our previous method [9], an aliquot of hippocampal homogenate in 0.4 M Tris-buffer (pH 8.5) was centrifuged at 4 • C to obtain the supernatant as a DAAO source for both the KYNA assay and hydrogen peroxide detection.
KYNA assay: Hippocampal homogenate supernatant (35 µL of 5-8 µg/µL) was incubated with FAD solution (2 mM, 5 µL) for 5 min at 37 • C. Subsequently, D-kynurenine (10 mM, 10 µL) in 0.4 M Tris-buffer (pH 8.5) was added to prepare the mixture for metabolizing D-kynurenine at 37 • C for 3 h. The DAAO enzymatic reaction was terminated by adding 50% trichloroacetic acid (5 µL). After mixing and centrifugation, the supernatant of the reaction solution (50 µL) and zinc sulfate (300 mM, 20 µL in ddH 2 O) were added to 300 µL of 0.4 M Tris-buffer (pH 8.5) for the final fluorescence assay (excitation wavelength of 251 nm and emission wavelength of 396 nm) with an EnSpire 2300 Multimode Plate Reader (Perkinelmer Co., Waltham, MA, USA). The DA (DAAO activity) was calculated as K/M/T (K = A × 370 µL, where A is amount of KYNA according to the standard treatment of the relationship between the optical density and concentration of KYNA; M is the mass of the total protein and is equal to the concentration of total protein × 35 µL; and T is the duration of the reaction, where T = 3 h × 60 = 180 min). The final DAAO activity was indicated in the form of the production rate of KYNA (fmol KYNA/µg protein/min) after calibration of the standard curve.
Hydrogen peroxide detection: Hippocampal homogenate supernatant and astrocyteconditioned medium were tested using an aqueous hydrogen peroxide assay kit (Sangon Biotech, Shanghai, China). In brief, samples of supernatant diluted by 20 times were incubated with the reaction solution at room temperature for 0.5-1 h. The OD 590nm values were recorded using a Varioskan Flash spectral reader (Termo Labsystems, Philadelphia, PA, USA). The hydrogen peroxide levels were normalized and quantified on the basis of a standard curve [1].

TdT-Mediated dUTP Nick-End Labeling (TUNEL) and Nissl Staining
Coronal sections of the hippocampus were prepared as mentioned in Section 2.5. Coronal sections (30 µm thickness) were first prepared by deparaffinizing and rehydrating before staining in toluidine blue solution for 30 min at 60 • C. Then, slides were washed with distilled water and ethanol gradients (70%, 80%, 95%, and 100%) for 1 min in succession. Pure dimethylbenzene was added to enhance light transmission, and coverslips were fixed with neutral balsam. In subsequent applications of an optical microscope, numbers of total neurons (400× field) in the hippocampal CA2 region were counted and calculated blindly by an investigator. TUNEL immunostaining was studied according to the instructions of the TUNEL cell apoptosis detection kit (Beyotime Institute of Biotechnology, Nantong, China). For Nissl staining, the percentage of injured cells = number of injured cells/(number of injured cells + number of normal neurons) × 100.

Isolation of Primary Cells
After fixing brains of one-day-old postnatal pups, the meninge was quickly peeled off using ophthalmic tweezers under a low-power inverted microscope on an ultra-clean platform. One side of the cerebrum was everted gently from the longitudinal fissure near the upper cerebellum. A large number of ependymal attachments were observed near the cerebellum; then, the hippocampus could be recognized and was immediately separated. Mouse hippocampi were minced and digested with 0.05% trypsin (Beyotime Institute of Biotechnology, Nantong, China) for 0.5 h. After screening with 70 and 40 µm filters, dispersive cells were neutralized with 5 mL of complete Dulbecco's modified Eagle's medium (Gibco DMEM, Life Science Inc, Woodland Hill, CA, USA) containing 10% fetal bovine serum (FBS, v/v) and 1% penicillin/streptomycin and centrifuged (600× g) for 5 min before resuspension. The cellular suspension was seeded in a 100 µg/mL poly-L-lysine-coated cell culture flask (1 × 10 6-7 ) and co-cultured for 10 days. Confluent cultures were shaken at 1× g overnight, and culture medium was changed to remove microglia and neurons. DMEM was used to wash culture flasks and remove unattached cells. Oligodendrocytes were detached with 0.05% trypsin at 37 • C for 15 min, and the suspension was eliminated. The remaining purified astrocytes were cultured in complete culture medium and plated for experiments.
For the collection of primary neurons, suspended cells collected by shaking confluent cultures overnight were incubated in complete DMEM for 2 h. Then, neurobasal medium (Invitrogen, Carlsbad, CA, USA) containing Gibco B-27 supplement (50×, Life Science Inc., Woodland Hill, CA, USA) and glutamine (0.5 mM, Life Science Inc, Woodland Hill, CA, USA) was used to perform selective neuronal culture for 5 days. In this neurobasal medium, glial cells were inhibited, and the remaining cells were identified as purified neurons.

Construction of pcDNA3.1(+)-daao and Transfection in Primary Astrocytes
The method in detail was performed as in previous reports [16,17]. daao cDNA was obtained from the hippocampal total mRNA of Swiss mice (MagExtractor-RNA kit, Toyobo Co., Osaka, Japan) after reverse transcription using a ReverTra Ace qPCR RT-Kit (Toyobo Co., Osaka, Japan) and was amplified using the following primers: Hindaao-F: 5 -TTTAAGCTTatgcgcgtggccgtgatc-3 and Ecodaao-R: 5 -TTTGAATTCtcagaggtgggagggaggcaac-3 . Then, the daao coding sequence was introduced into the HindIII and EcoRI multicloning sites of expression vector pcDNA3.1. The transcription of daao was driven by CMV promoter. After culturing for 6 days, primary hippocampal astrocytes were transfected with 60 nM daao cDNA cloned in pcDNA3.1 or empty vectors (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using LipoMax transfection reagent (Sudgen, Nanjing, China) and cultured at 37 • C for 2 days. The daao mRNA levels and DAAO protein expression were confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot, as mentioned above. pcDNA3.1(+)-daao-transfected primary astrocytes were utilized to mimic the pathological overexpression of astrocytic DAAO. Astrocyte-conditioned medium (ACM) was transferred into culture plates seeded with primary neurons to explore causal relationships with delayed neuronal death and potential mechanisms.

Apoptosis, Cell Viability, and Lactate Dehydrogenase (LDH) Cytotoxicity Assay
After D-Ala (0.3 mM or 1.0 mM) and 2.5 µM FAD were added in culture media with pcDNA3.1-transfected astrocytes and incubated for 6 h, ACM was transferred into culture plates seeded with primary neurons for subsequent studies. For the apoptosis study, primary neurons (1 × 10 5 ) were seeded into a 100 µg/mL poly-L-lysine-coated 96-well plate for 4-5 days at 37 • C in a 5% carbon dioxide incubator and incubated with ACM for 2 or 4 days. ACM was renewed every 24 h, and Gibco B-27 supplement (50×, Life Science Inc, Woodland Hill, USA) and glutamine (0.5 mM, Life Science Inc, Woodland Hill, CA, USA) were added to ACM to support neuronal growth. Apoptosis was evaluated using a TUNEL fluorescent probe detection kit (Beyotime Institute of Biotechnology, Nantong, China). Images were obtained with a fluorescence microscope (Nikon Instruments Inc., Melville, NY, USA) at 100× magnification. To quantify the percentage of apoptotic neurons, photos were captured for five random fields of each well, and TUNEL-positive cells were counted and averaged.
In addition, a CCK-8 assay kit (Beyotime Institute of Biotechnology, Nantong, China) was applied to measure the level of yellow formazan dye produced by dehydrogenase in the living cells for cell viability. A portion of neurons (5 × 10 3 cells/mL) was inoculated with complete DMEM in 96-well plates and maintained in an incubator at 37 • C for one day. Subsequently, the culture medium was replaced with ACM, and neurons were co-cultured with other chemicals (50 µM SUN compound, 10 µM SKF96365, or 10 µM PBN) for one day at 37 • C. Lastly, CCK-8 reagent was incubated with neurons in each well at 37 • C for 2-3 h. OD 450nm values were detected with a Multimode Plate Reader (EnSpire 2300, Perkinelmer Co., Waltham, MA, USA).
LDH cytotoxicity was measured using an LDH assay kit following the manufacturer's instructions (Beyotime Institute of Biotechnology, Nantong, China). After incubation with ACM and other chemicals for one day, the medium was removed and replaced with 150 µL PBS-diluted LDH release reagent. The mixture was shaken gently for 10 s and incubated in a cell incubator for 1 h at 37 • C. After shaking at 500× g for 5 min, 150 µL of supernatant was transferred to a new 96-well plate for determination. The test was based on the content of LDH released into the culture medium by damaged neurons. Absorbance at the wavelength of 490 nm was recorded with a Multimode Plate Reader (EnSpire 2300, Perkinelmer Co., Waltham, MA, USA). The results were presented in the form of percentages by calculating the contents of LDH released from neurons in different groups compared to the control group: cell cytotoxicity (%) = (OD test group − OD control group )/(OD test group − OD blank ) × 100%.

Statistical Analysis
Shapiro-Wilk normality testing was conducted to evaluate the distribution of data. Data following non-normal distributions are presented in the form of medians and interquartile ranges and were analyzed via a nonparametric equivalent. One-way ANOVA with a post hoc Student-Newman-Keuls test was applied for time-course-related experiments, TUNEL-labeled apoptotic tests, cell activity assays, and cytotoxicity assays. Unpaired two-tailed Student's t-test was used for the evaluation of DAAO expression in pcDNA3.1(+)-daao-transfected astrocytes as well as treatment-related experiments for the prevention of neurobehavioral deficits and delayed hippocampal neuronal death after MCAO surgery. Normally distributed data are presented as means ± SE and analyzed using GraphPad Prism 7 (GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was defined as a p-value less than 0.05.

Cell Specificity of Hippocampal DAAO Expression in Ischemic Mice Induced by Transient MCAO
Considering that both microglia and astrocytes are activated and sustained for many days in ischemic strokes [19,20], the timepoint of day 3 after surgery was used to clarify the cell specificity of hippocampal DAAO in ischemic mice by immunocolocalizing with Iba-1, NeuN, or GFAP, respectively. As shown in the photomicrographs, DAAO-positive cells were extensively labeled with GFAP, whereas no or few DAAO-positive cells were colabeled with Iba-1 or NeuN ( Figure 1A-C). The area fractions of DAAO colocalizing with three cellular markers are quantified in Figure 1D. The results suggest that hippocampal DAAO is mainly expressed in activated astrocytes in ischemic stroke associated with complex morphological changes, including cellular hypertrophy. Subsequently, immunocolocalization of double-labeled GFAP/DAAO was also quantified at four timepoints (0 h, 24 h, 3 days, and 5 days) following MCAO surgery using a computer-assisted image analysis program (0.025 ± 0.003%, 0.075 ± 0.013%, 2.76 ± 0.28%, and 3.65 ± 0.20%, respectively) ( Figure 1E-P).

Time Course of Hippocampal DAAO Expression and Related Activity after Acute Ischemic Insult
Western blotting was employed first to disclose hippocampal DAAO expression following MCAO injury (relative to β-actin). Mice in four groups (0 h, 24 h, 3 days, and 5 days) were euthanized on the same day to prepare hippocampal samples of all the section slices. Hippocampal DAAO expression was significantly increased by 0.38 times, 0.64 times, and 1.1 times at 24 h (p = 0.08), 3 days (p = 0.008), and 5 days (p = 0.0003), respectively, following MCAO surgery compared to the sham group, as calculated by one-way ANOVA with post hoc Student-Newman-Keuls test (F(3, 16) = 13.4, p = 0.0001, Figure 2A). Double-labeled nestin/DAAO as well as GFAP/DAAO/DAPI immunostaining were applied to study DAAO expression in hippocampal astrocytes at different timepoints after ischemic insult. Of note, nestin is a much more specific marker for reactive astrocytes, and the quantification of nestin/DAAO colocalization is critical to reveal expression of hippocampal DAAO in activated astrocytes. Double-labeled nestin/DAAO immunocolocalization was quantified at four timepoints (0 h, 24 h, 3 days, and 5 days) following MCAO surgery with a computer-assisted image analysis program (F(3, 16) = 204.5, p < 0.0001; 0.74 ± 0.06%, 1.61 ± 0.09%, 7.12 ± 0.43%, and 8.88 ± 0.35%, respectively). As shown in Figure 2B-N, the intensity of nestin/DAAO was strikingly increased by more than 11.0 times on day 5 after MCAO onset (p = 0.0001, as calculated by one-way ANOVA with post hoc Student-Newman-Keuls test).

Time Courses of Delayed Hippocampal Neuronal Death in Ischemic Stroke
Neurobehavioral deficit scoring was performed first to evaluate MCAO-induc cerebral damage at four timepoints (0 h, 24 h, 3 days, and 5 days) following MCAO su gery ( Figure 3A; F (3, 20) = 34.72, p < 0.0001; 0.17 ± 0.17, 1.83 ± 0.31, 2.82 ± 0.31, and 3.67 0.21, respectively). Intravenous, twice-daily injections 4H-furo[3,2-b]pyrrole-5-carboxylic acid DAAO inhibitor (SUN, 10 mg/kg) notably inhi ited behavioral deficits on day 5 after MCAO onset by 38.0% compared to the mod group treated with saline (t = 2.90, p = 0.016, as calculated by unpaired two-tailed St dent's t-test; Figure 3B). This experiment confirmed previously established data th SUN reduces neurological deficits following MCAO, but it was assessed using a diffe ent system for scoring. The immunostaining colocalization of hippocampal GFAP/DAAO was perform compared with mice at the timepoint of 0 h or in the sham group; # denotes significant difference from model group treated with saline (p < 0.05) calculated by one-way ANOVA with post hoc Student-Newman-Keuls test or unpaired two-tailed Student's t-test (nonparametric statistics for neurological scores).
The immunostaining colocalization of hippocampal GFAP/DAAO was performed on day 5 in MCAO mice. The results indicate that DAAO was mainly expressed in GFAPpositive astrocytes in the hippocampal sections ( Figure 4A-D). In addition, hippocampal immunofluorescence with NEUN and TUNEL staining was executed ( Figure 4E-H). The results show that apoptotic neurons are also mainly localized in the hippocampal CA2 area. Thus, the activation of astrocytic DAAO during ischemic stroke might contribute to neuronal death. In the Nissl-staining study, the numbers of normal neurons and injured cells in the hippocampal CA2 area were recorded on day 5 after ischemic stroke ( Figure 4I-K). Hippocampal neurons containing Nissl bodies were recorded as normal cells. Pyknotic nuclei with condensed chromatin were defined as injured cells. The number of total neurons in the model group treated with saline was deceased by 29.7% compared to the sham group, which was alleviated by the administration of SUN (10 mg/kg) at an elevation of 17.3% (t = 3.96, p = 0.0027, as calculated by unpaired two-tailed Student's t-test; Figure 4L). In particular, in the analysis of injured cells, injured neurons were significantly decreased in the treatment group by 82.7% compared to model rats treated with saline (t = 37.3, p = 0.0001, as calculated by unpaired two-tailed Student's t-test; Figure 4M).

Construction of pcDNA3.1(+)-daao in Primary Astrocytes and Apoptotic Test
To further confirm the role of hippocampal DAAO in MCAO-induced delayed neuronal death, pcDNA3.1(+)-daao-transfected primary astrocytes were used to mimic the impact of DAAO overexpression on hippocampal neuronal death in an ischemia-reperfusion animal model. RT-qPCR and Western blot technology were applied to evaluate the mRNA and protein levels of DAAO in pcDNA3.1(+)-daao-transfected primary astrocytes. DAAO was overexpressed in transfected primary astrocytes compared to both normal astrocytes and blank pcDNA3.1(+)-transfected astrocytes ( Figure 5A,B) Cells 2022, 11, x FOR PEER REVIEW 12 of 18 two-tailed Student's t-test; Figure 4L). In particular, in the analysis of injured cells, injured neurons were significantly decreased in the treatment group by 82.7% compared to model rats treated with saline (t = 37.3, p = 0.0001, as calculated by unpaired two-tailed Student's t-test; Figure 4M). . Data are expressed as means ± SEM (n = 6). * denotes statistical significance (p < 0.05) compared with sham group; # denotes significant difference from the model group treated with saline (p < 0.05) calculated by unpaired two-tailed Student's t-test.
FAD is an essential cofactor for the catalytic activity of DAAO; thus, it should be added to all tests of DAAO activity. After incubating D-Ala (0.3 mM or 1.0 mM) and 2.5 µM FAD with pcDNA3.1-transfected astrocytes for 6 h, the ACM was transferred to a 96-well plate seeded with primary neurons (1 × 10 5 ) for apoptotic testing. The apoptotic status of the hippocampal neurons was determined using a fluorescent TUNEL assay kit, with the greatest increases of 1.7 times and 2.2 times on day 2 and day 4, respectively, after incubation in ACM containing 1.0 mM D-Ala (p = 0.012 and p = 0.0034, respectively, calculated by oneway ANOVA with post hoc Student-Newman-Keuls test; Figure 5C-M). TUNEL-positive cells were also increased by 60.3% and 86.4% on days 2 and 4, respectively, after incubation in ACM containing 0.3 mM D-Ala (p = 0.013 and p = 0.0016, respectively). The levels of H 2 O 2 , as the byproduct of D-Ala, were also measured in pcDNA3.1(+)-daao-transfected astrocytes treated with different D-Ala concentrations (0.03 mM, 0.3 mM, and 1.0 mM) after enzymatic reaction for 30 min ( Figure 5N). These results imply that DAAO-induced neuronal apoptosis might be associated with byproduct hydrogen peroxide through the enzymatic reaction of the D-Ala substrate.

Involvement of Transient Receptor Potential (TRP) Calcium Channels and H 2 O 2 in DAAO-Induced Neuronal Apoptosis
ROS (including H 2 O 2 ) directly activate TRP calcium channels (such as TRPA1, TRPV1, TRPM2, TRPC5, etc.), promote calcium influx [21,22], aggravate mitochondrial calcium ion overloading [23], and induce neuronal apoptosis [24,25]. Therefore, many drugs have been employed to explore the potential mechanism of ACM-induced neuronal apoptosis. DAAO inhibitor SUN (50 µM), nonselective TRP calcium channel antagonist SKF96365 (10 µM), and active oxygen scavenger PBN (10 µM) were added to transfected astrocytes with substrate D-Ala and incubated for one day. Different ACM types from treatment subgroups showed diverse improvements in cell viability and cytotoxicity in the sequence of PBN > SUN > SKF96365, as calculated using one-way ANOVA with post hoc Student-Newman-Keuls test ( Figure 6A,B). The results demonstrate that TRP calcium channels and H 2 O 2 could be involved in DAAO-induced neuronal apoptosis.
Cells 2022, 11, x FOR PEER REVIEW 15 of calcium ion overloading [23], and induce neuronal apoptosis [24,25]. Therefore, ma drugs have been employed to explore the potential mechanism of ACM-induced ne ronal apoptosis. DAAO inhibitor SUN (50 μM), nonselective TRP calcium channel a tagonist SKF96365 (10 μM), and active oxygen scavenger PBN (10 μM) were added transfected astrocytes with substrate D-Ala and incubated for one day. Different AC types from treatment subgroups showed diverse improvements in cell viability and c totoxicity in the sequence of PBN > SUN > SKF96365, as calculated using one-w ANOVA with post hoc Student-Newman-Keuls test ( Figure 6A,B). The results demo strate that TRP calcium channels and H2O2 could be involved in DAAO-induced ne ronal apoptosis.

Discussion
DAAO is traditionally recognized as a glial enzyme localized in the peroxisome astrocytes and Bergmann glia [26,27]. The results presented in this study confirm th hippocampal DAAO is specifically expressed in reactive astrocytes and only barely in t neurons of ischemia-reperfusion mice. In our earlier study, we found that an ischem insult strikingly stimulates astrocytes in the cortical peri-infarct area, resulting in hyp trophy or hyperplasia, which results in formation of a cellular barrier, and DAAO w specifically expressed in reactive astrocytes [9]. Cerebral infarctions and neurobehavio deficits induced by transient MCAO deteriorated over time, particularly on day 5 af surgery, which was related in parallel to a continual increase in DAAO expression. W therefore suppose that there might be a causal relationship between the high expressi of DAAO and cerebral damage in ischemia-reperfusion mice. In this study, an identic situation was also demonstrated in hippocampal sections. The intravenous injection DAAO inhibitor SUN distinctly reversed delayed hippocampal neuronal death in MCA mice. Thus, it appears that high expression of DAAO might be also related to delay neuronal death after ischemic insult. We found that the DAAO inhibitor SUN marked relieves the degradation in behavioral status and delays hippocampal neuronal death ischemic mice.

Discussion
DAAO is traditionally recognized as a glial enzyme localized in the peroxisome of astrocytes and Bergmann glia [26,27]. The results presented in this study confirm that hippocampal DAAO is specifically expressed in reactive astrocytes and only barely in the neurons of ischemia-reperfusion mice. In our earlier study, we found that an ischemic insult strikingly stimulates astrocytes in the cortical peri-infarct area, resulting in hypertrophy or hyperplasia, which results in formation of a cellular barrier, and DAAO was specifically expressed in reactive astrocytes [9]. Cerebral infarctions and neurobehavioral deficits induced by transient MCAO deteriorated over time, particularly on day 5 after surgery, which was related in parallel to a continual increase in DAAO expression. We therefore suppose that there might be a causal relationship between the high expression of DAAO and cerebral damage in ischemia-reperfusion mice. In this study, an identical situation was also demonstrated in hippocampal sections. The intravenous injection of DAAO inhibitor SUN distinctly reversed delayed hippocampal neuronal death in MCAO mice.
Thus, it appears that high expression of DAAO might be also related to delayed neuronal death after ischemic insult. We found that the DAAO inhibitor SUN markedly relieves the degradation in behavioral status and delays hippocampal neuronal death in ischemic mice.
In transfected primary astrocytes, the overexpression of DAAO accompanying astrocytic activation is highly cytotoxic due to the byproduct hydrogen peroxide, which is a stable reactive oxygen species, and significant neuronal apoptosis and necrosis occur [28]. DAAO-mediated hyperalgesia, morphine nociceptive tolerance, and neurodegenerative damage are speculated to be associated with hydrogen peroxide [1,2,24]. Our previous results demonstrated that the DAAO inhibitor SUN distinctly diminishes the whole-brain levels of hydrogen peroxide [9]. Thus, we postulated that the DAAO/hydrogen peroxide pathway is at least partly responsible for delayed neuronal death and neurobehavioral deficits. To further explore direct evidence for the relationship between delayed neuronal death and DAAO overexpression, an in vitro DAAO overexpression model was applied by transfecting the target plasmid into primary astrocytes, and ACM was used for incubation with primary neurons to induce apoptosis. Meanwhile, our results indicated that both TRP calcium channels and H 2 O 2 are involved in DAAO-induced neuronal apoptosis. H 2 O 2 can cause astrocytic metabolism disorder, induce an increase in active oxygen free radicals, and decrease BDNF expression [10,11]. Hence, it is supposed that the high level of ROS and reduced BDNF in ACM might elicit apoptosis in neuronal cells. ROS (including H 2 O 2 ) directly activate TRP calcium channels (such as TRPA1, TRPV1, TRPM2, TRPC5, etc.), promote calcium influx [21,22], and aggravate mitochondrial calcium ion overloading [23], resulting in the downregulation of a variety of synaptic protein expression and neuronal apoptosis [24,25]. In addition, the low levels of BDNF in ACM might inactivate the functions of tyrosine kinase receptor B in cellular survival and synaptic plasticity via phosphoinositide 3-kinase/protein kinase B, mitogen-activated protein kinase, etc. [29,30].
A few limitations of this study need to be reported. We did not set up a sham group as a part of the study design, which is a limitation of this study. Our outcomes showed that DAAO and its substrate D-Ala only promoted neuronal apoptosis in a dose-dependent manner. Whether DAAO overexpression in neuropathological disorders could also exhibit cytotoxicity is undefined. The influence of inhibiting DAAO on learning ability and memory should also be confirmed in further work.
In conclusion, we found that astrocytic activation induced by ischemic insult is concomitant with the increasing expression of DAAO over a time period of several days. In addition, the current outcomes suggest that the high expression of hippocampal DAAO is causally associated with delayed neuronal death at subacute phases of ischemiareperfusion, at least partly, by enhancing H 2 O 2 levels with subsequent activation of TRP calcium channels in neurons. This study demonstrated the involvement of DAAO in astrocyte-neuron interactions in neuropathological disorders as well as that the mechanism underlying neuronal apoptosis is induction by astrocytic DAAO. DAAO could thus serve as a therapeutic target in the management of ischemic stroke and other neurodegenerative diseases.

Institutional Review Board Statement:
A statement for humans is not applicable since there were no human subjects or samples in this study. Animal protocols were approved by the Animal Care and Welfare Committee of Shanghai Jiao Tong University (Shanghai, China) and were performed in accordance with the Guide for Care and Use of Laboratory Animals and the Animals in Research: Reporting In Vivo Experiments guidelines.

Informed Consent Statement: Not applicable.
Data Availability Statement: All the data generated or analyzed during this study are included in the published article.


Angiogenin promotes angiogenesis via the endonucleolytic decay of miR-141 in colorectal cancer

Mature microRNA (miRNA) decay is a key step in miRNA turnover and gene expression regulation. Angiogenin (ANG), the first human tumor-derived angiogenic protein and also a member of the RNase A superfamily, can promote tumor growth and metastasis by regulating rRNA biogenesis and tiRNA production. However, its effect on miRNA has not been explored. In this study, we find that ANG exclusively downregulates mature miR-141 in human umbilical endothelial cells (HUVECs) via its ribonuclease activity and preferably cleaves single-stranded miR-141 at the A5/C6, U7/G8, and U14/A15 sites via endonucleolytic digestion. By downregulating miR-141, ANG promotes HUVECs proliferation, migration, tube formation, and angiogenesis both in vitro and in vivo. Conversely, downregulated ANG inhibits ANG-mediated miR-141 decay, thus decreasing the angiogenesis process of HUVECs. We also find an inverse correlation between ANG and miR-141 expression in colorectal cancer (CRC) tissues. Our study indicates that ANG regulates CRC progression by disrupting miR-141 and its regulation on angiogenesis-related target genes, not only revealing a new mechanism of ANG action but also newly identifying miR-141 as a substrate of ANG. This study suggests that targeting ANG nuclease activity might be valuable in treating angiogenesis-related diseases through coordinately regulating the metabolism of rRNA, tiRNA, and miRNA.

INTRODUCTION
Angiogenin (ANG), the first human tumor-derived angiogenic protein, was originally isolated from the conditioned medium of HT-29 human colon adenocarcinoma cells based solely on its angiogenic activity. 1 It has now been found to promote tumor growth and metastasis, 2,3 exhibit microbicidal activity in innate immunity, 4 enhance neuron outgrowth and survival, 2,5 differentially regulate stem and progenitor cell quiescence, 6 and protect intestinal epithelial barrier integrity 7 and shape the gut microbe composition 8 to maintain gut health. However, because of the presence of multiple homologs in model organisms, its physiological and pathological roles and mechanisms of action have not yet been fully elucidated.
ANG is the fifth member of the RNase A superfamily. Although its ribonucleolytic activity is only 10 À5 -to 10 À6 -fold of what is in RNase A, this activity is essential for ANG to perform its biological functions. 2 Loss-of-function mutations in the catalytic sites of ANG have been documented in neurodegenerative diseases, such as amyotrophic lateral sclerosis and Parkinson's disease. 2,9,10 It has long been speculated that certain cellular RNAs can be processed as substrates of ANG, which has been supported by studies on rRNAs and tRNAs. [11][12][13][14] In the growth state, ANG both enhances rRNA transcription and participates in 47S pre-rRNA processing via endonucleolytic cleavage at the 5 0 external transcription spacer (A 0 site) to promote cell proliferation. 11 However, when under stress, ANG cleaves the anticodon loop of tRNAs to produce tRNA-derived, stress-induced small RNAs (tiRNAs) to maintain cell survival. [12][13][14] Therefore, exploring whether ANG can target other RNA subsets as substrates remains of general interest.
Angiogenesis occurs as an orderly cascade of molecular and cellular events that mainly include angiogenic factor secretion; receptor activation and signal transduction; and endothelial cell survival, proliferation, sprouting, migration, tube formation, and vascular stabilization; 15 thus, angiogenesis plays essential roles in several physiological and pathological processes, including embryonic development, wound healing, inflammation, and tumor growth and metastasis. 16,17 The role of ANG, besides enhancing rRNA biogenesis, in angiogenesis has been well established. ANG can also activate extracellular signal-related kinase 1/2 and protein kinase B/Akt in endothelial cells to produce more ribosomal proteins, thereby promoting cell growth. 18,19 ANG induces the synthesis of nitric oxide, a well-known regulator involved in vascular physiology, through the PI3K/Akt kinase signaling transduction cascade in endothelial cells. 20 In addition, ANG degrades the extracellular matrix and basement membrane to promote endothelial cell invasion and migration into the surrounding tissue. 21 MicroRNAs (miRNAs) are a class of highly conserved non-coding small RNAs that regulate the expression of target genes post-transcriptionally through translational repression or mRNA degradation. 22 Accumulating evidence suggests that miRNAs play critical roles in various bioprocesses, including angiogenesis and tumor progression. 23,24 To explore the mechanism of action of ANG in angiogenesis and miRNA metabolism, we created a model using human umbilical endothelial cells (HUVECs) and screened for ANG-responsive miR-NAs using an miRNA array. We identified miR-141 as one of the most downregulated miRNAs. Coincidentally, our previous results identified miR-141 as a critical angiogenesis-inhibiting miRNA. 25 In this study, we first confirmed that ANG downregulated mature miR-141 in HUVECs and then demonstrated that the underlying mechanism for this was endonucleolytic digestion of mature miR-141. We further established the relationship among ANG, miR-141 degradation, and angiogenesis both in vitro and in vivo. Finally, we provided evidence supporting the roles of ANG-miR-141-targets in tumor angiogenesis and the progression of human colorectal cancer (CRC).

ANG regulates miRNA expression in HUVECs
We adopted HUVECs as the model system to screen differentially expressed miRNAs in responding to recombinant human ANG protein treatment. As the immunofluorescence staining indicated, ANG was internalized and assembled in the cytoplasm and nucleus of the HUVECs ( Figure 1A). The miRNA expression profile assay indicated that 25 miRNAs were altered in response to ANG stimulation, including 9 downregulated and 16 upregulated miRNAs with a cutoff fold change of 0.5-or 2-fold compared with the control, respectively ( Figure 1B). Of these miRNAs, miR-141 (miR-141-3p) displayed the most striking response to ANG stimulation. We then confirmed the regulation of ANG on miR-141 by quantitative PCR and showed that the miR-141 levels decreased after ANG treatment ( Figure 1C) but increased following ANG knockdown in HUVECs ( Figure 1D). Consistently, we found that ANG regulated miR-141 levels in the CRC cell line SW480 ( Figure S1).

ANG specifically downregulates mature miR-141 in HUVECs
Canonical miRNA biogenesis is a multistep process that involves transcription, processing by Drosha and Dicer, RNA modification, nuclear export, the formation of miRISC (miRNA-induced silencing complex), and RNA decay. 22 ANG has been reported to regulate rDNA gene transcription via direct DNA binding, cleave pre-rRNA and tRNA via its ribonucleolytic activity, and modulate various biological processes via interactions with different proteins. 2 However, its role in miRNA biogenesis remains unknown. To explore whether ANG affects miRNA processing, we measured the amounts of pri-, pre-, and mature miR-141 (miR-141-3p) in HUVECs after treatment with recombinant ANG protein, the depletion of endogenous ANG by siRNAs, or ectopic overexpression of ANG with lentivirus. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses revealed that ANG only regulated mature miR-141 levels but did not change the pri-or pre-miR-141 levels ( Figures 1E-1G), suggesting that ANG does not modulate either pri-miR-141 gene transcription or pre-miR-141 processing. miRNA* sequences, which are the fragments from the opposing arm of miRNAs, are typically found at much lower frequencies than the miRNAs. 23 Although miR-141-5p (miR-141*) could not be detected in HUVECs, it could be detected but not regulated by ANG in SW480 cells ( Figure S2), suggesting the specificity of ANG's regulation on miR-141.
MiR-141 belongs to the miR-200 family, which includes miR-200a, miR-200b, miR-200c, miR-429, and miR-141. All of these members have very similar sequences, with differences of only several nucleotides. Moreover, miR-200c is located in the same cluster as miR-141, suggesting that they may share the same transcription cassette ( Figure S3A). To determine whether ANG affects the levels of other members of the miR-200 family, we also checked the expressions of these four members after ANG was manipulated and found that there were no significant changes, indicating that the regulation of ANG toward miR-141 is specific ( Figures S3B and S3C).
The regulation of ANG on miR-141 depends on its ribonuclease activity Given that ANG is a weak ribonuclease and must enter cells to exert its role, 2 we examined the contribution of its intracellular enzymatic activity to the downregulation of miR-141. When we treated the cells with RNase inhibitor (RI) or the ANG inhibitor neomycin (Neo) 26 to block ANG's activity, ANG's role in regulating miR-141 nearly vanished ( Figure 1J). K40 and H114 are two key amino acids in the catalytic center of ANG, and their respective mutants of ANG-K40I and ANG-H114A exhibited null or little RNA hydrolase activity in vitro ( Figure 1K). 27 When we treated HUVECs with either of these two mutants, the downregulation of mature miR-141 was completely abolished ( Figure 1L). To determine whether this phenomenon is ANG-specific, we treated the cells with RNase 4 or RNase A, which are two other members of the RNase A family with similar or much stronger enzyme activity compared with ANG ( Figure 1K). The results clearly showed that neither RNase was able to downregulate miR-141 in HUVECs ( Figure 1L).

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We performed an RNA-binding protein immunoprecipitation (RIP) assay with anti-ANG antibodies to explore whether ANG cleaves miR-141 through a direct interaction in cells. The immunoprecipitated RNAs were then purified and analyzed by qRT-PCR and semi-quantitative PCR. The results revealed that only mature miR-141 was enriched by ANG, with no changes in U6, pre-miR-141, pri-miR-141, or GAPDH mRNA (Figures 2A-2C). To more clearly dissect the localization and interaction between ANG and miR-141, we labeled miR-141 with Cy5 either at the 3 0 end or the 5 0 end and used immunofluorescence to visualize ANG. The image revealed obvious co-localization of Cy5-miR-141 with ANG in the cytoplasm in SW480 and HUVEC ( Figures 2D and S4). To confirm these observations, we further detected pre-and mature miR-141 levels by northern blotting. The results showed that mature miR-141 increased when ANG was knocked down, whereas pre-miR-141 remained at almost the same levels as those of the control group ( Figures 2E and 2F). When the cells were infected with the lentivirus to overexpress ANG or ANG-K40I mutant, mature miR-141 decreased only in the ANG-overexpressing cells ( Figures 2G and  2H). These results demonstrated that ANG interacts with miR-141 in the cytoplasm and regulates the level of mature miR-141 through its ribonuclease activity.

ANG cleaves single-stranded miR-141 as an endonuclease in vitro
To explore the biochemical characteristics and cleavage pattern of ANG in mature miR-141, we developed a cell-free cleavage assay with synthetic miR-141 analogs or its variated forms, including wildtype single-stranded mature miR-141, a single-stranded DNA oligonucleotide with the same sequence of miR-141 (DNA-ss-miR-141), an miR-141 RNA duplex with complete complementary duplex, an miR-141 mimic with complementary duplex with 2-nucleotide 3 0 overhangs, and a single-stranded miR-141 with different modifications (methylation or uridylation). The results showed that the synthetic single-stranded miR-141 could be successfully degraded by ANG but not by ANG mutants without ribonuclease activity ( Figure 3A). ANG did not digest the DNA sequence of the miR-141 version ( Figure 3B) or the miR-141 duplex ( Figure 3C). ANG only digested the 3 0 end overhanging 2-nucleotide of the miR-141 mimic ( Figure 3D). Furthermore, the methylated nucleotides were resistant to ANG digestion. When miR-141 was modified with whole-strand nucleotide 2 0 -O-methyl-ation, ANG could no longer digest miR-141 ( Figure 3E); however, ANG could still cleave the single-stranded miR-141 with 2 0 -O-methylation at both the 3 0 and 5 0 ends ( Figure 3F), clearly indicating that ANG cleaves miR-141 as an endonuclease in vitro and that nucleotide modified with 2 0 -O-methylation protects RNA from ANG digestion. Meanwhile, uridylation at the 3 0 end of single-stranded miR-141 could accelerate ANG digestion on miR-141 to a certain extent (Figures 3G and 3H please compare with Figure 4A). The electrophoretic mobility shift assay (EMSA) results showed that recombinant ANG interacts with Cy5-miR-141 during cleavage while the mutant ANG-K40I is unable to perform this action ( Figure 4I). The interaction of ANG/Cy5-miR-141 could be blocked by RI (Figure 4J) or competitively inhibited by unlabeled miR-141 ( Figure 4K). Further analysis revealed that most of the substrate (3.57 pmol/mL) was degraded by ANG (0.71 pmol/mL) in 30 min, indicating that one molecule of ANG is sufficient to cleave five molecules of mature miR-141 in 30 min ( Figure 4A).
ANG cleaves single-stranded miR-141 at the A 5 C 6 , U 7 G 8 , and U 14 A 15 sites We next analyzed the digestion pattern to investigate the cleavage details of ANG on miR-141. Generally, there were three major RNA fragment bands in the SYBR Gold dye-stained gel, corresponding to 17, 15, and 14 nt in length ( Figure 4A). Based on the band length and the knowledge that ANG preferentially catalyzes RNA after pyrimidine, 28 we synthesized a series of mutated miR-141s at certain or all U sites and the 5A site ( Figure 4B). As the results indicate in Figure 4, when more than four Us were replaced by Gs, ANG completely failed to cleave these miR-141 derivatives ( Figures 4C and 4D). When the 7th and 14th U were mutated to G, two major digestion product bands with lengths of 15 and 14 nt were lost ( Figure 4E). When one of the three sites of 5A, 7U, and 14U was replaced, one corresponding cleavage product band was missed ( Figures 4F-4H).
Given that the SYBR Gold dye is not sensitive enough to stain smaller ribonucleotides, we labeled miR-141 with Cy5 at the 5 0 or 3 0 ends and then performed the cell-free cleavage assay to further analyze the cleavage site precisely. The data not only confirmed that ANG preferentially digested miR-141 at the A 5 /C 6 , U 7 /G 8 , and U 14 /A 15 sites but also indicated other feeble cleavage sites, such as U 11 /G 12 ( Figures 4I and 4J). www.moleculartherapy.org

ANG promotes angiogenesis by downregulating miR-141 in vitro and in vivo
In our previous study, we reported that miR-141 inhibits multiple angiogenesis-related biological processes, indicating that miR-141 plays a powerful and negative regulatory role in angiogenesis. 25 To underscore the biological significance of this specific ANG-mediated miR-141 decay, we want to establish the relationship between miR-141-inhibited angiogenesis and ANG-induced angiogenesis. Therefore, we manipulated the levels of ANG and miR-141 in HUVECs and evaluated the coordination of ANG and miR-141 in regulating angiogenesis processes. The results showed that the tube formation inhibition induced by miR-141 could be rescued by adding ANG back into the cell culture medium (Figures 5A-5C) or by overexpressing ANG in HUVECs ( Figure S5); this finding suggests that ANG promotes tube formation by downregulating miR-141. Considering that the downregulation of ANG could increase miR-141 levels by attenuating the cleavage effect of ANG on miR-141, we knocked down ANG using siRNAs in miR-141-depleted cells and found that the enhanced tube formation by the miR-141 inhibitor was significantly repressed again ( Figures 5D-5F). The reverse effect of ANG and miR-141 was also observed for HUVEC proliferation and migration ( Figures 5G and 5H).
To further validate the regulatory axis between ANG and miR-141 in angiogenesis, an in vivo Matrigel plug assay was employed. In accordance with the in vitro data, fewer vessels in the Matrigel plugs contained agomiR-141, but the vessels were much denser in the Matrigel plugs containing ANG than in the control group ( Figures 5I-5K). The combination of agomiR-141 with ANG resulted in a greater median number of vessels than that in the agomiR-141 group but a significantly smaller number than that in the ANG group ( Figures 5I-5K), indicating a mutual antagonism between ANG and miR-141 in angiogenic processes in vivo. Together, these results indicate that ANG promotes angiogenesis by downregulating miR-141 both in vitro and in vivo.

ANG, miR-141, target expression, and angiogenesis in colorectal cancer
Angiogenesis is a critical process for solid tumor growth and progression. ANG increases tumor growth while miR-141 has been shown to reduce microvessel density and primary tumor growth. 29 To validate our proposed regulatory axis in human samples, we measured the levels of ANG and miR-141 in plasma and tissues from CRC patients and observed a negative correlation between these values ( Figures 6A  and 6B). We then collected primary colorectal tumors and paired adjacent normal tissues from ten patients with CRC to further investigate the relationships among ANG, miR-141, miRNA-141 targets, and angiogenesis. Our results firstly confirmed the increased microvessel density in tumor tissues ( Figures 6C and 6D), and further measurement revealed that ANG mRNA expression was elevated and miR-141 expression was reduced in CRC tissues compared with their adjacent normal tissues ( Figures 6E and 6F). Moreover, in the four angiogenesis-related targets of miR-141, 25 we found that the protein levels of GATA6 and NRP1 were elevated in tumor tissues ( Figures   6G-6K), in accordance with the ANG protein level, which was shown in our previously published article. 3 In addition, in the paired CRC cell lines SW480 and SW620, which were established from a primary adenocarcinoma of the colon and a lymph node derived from the same patient, a consistent expression pattern was observed both in the cells and in their cultured medium (Figures 6L-6M).

DISCUSSION
miRNAs play critical roles in nearly all developmental and pathological processes. 22 Currently, its biogenesis has been well documented, but the understanding of miRNA turnover is quite limited, particularly in higher organisms. It is generally accepted that miRNA turnover contributes to its homeostasis and represents an important aspect of miRNA regulation. To date, several miRNases (miRNA-degrading nucleases) have been identified, including SDN, 30 XRN2, 31 and RRP6 32 in lower organisms, and XRN1, 33 hPNPase old-35 , 34 Eri1, 35 and TSN 36 in mammalian cells. Most of these miRNases are exoribonucleases. For example, XRN2 is a 5 0 -3 0 exoribonuclease, and SDN1, RRP6, XRN1, hPNPase old-35 , and Eri1 are 3 0 -5 0 exoribonucleases. Until now, TSN was the only endonuclease identified in mature miRNA turnover. Here, we report that ANG could specifically downregulate mature single-stranded miR-141 through endonucleolytic cleavage; both in vitro and in vivo studies demonstrated that the biological consequence of this downregulation is the induction of angiogenesis. Thus, we not only identified ANG as an miRNA turnover-related endonuclease, but also illustrated a novel mechanism in which ANG and miR-141 regulate angiogenesis.
Although ANG has been studied for more than 30 years, its physiological and/or pathological functions are not yet fully understood. 2 Very recently, this protein has been reported as a stem/progenitor cell regulator that plays dichotomous roles in reducing the proliferative capacity of primitive hematopoietic stem/progenitor cells (HSPCs) to maintain quiescence while simultaneously facilitating the proliferation of lineage-committed myeloid-restricted progenitor (MyePro) cells. 6 Mechanistically, this unique function is attributed to cell-type-specific RNA-processing events related to ANG, i.e., tiRNA generation in HSPCs and rRNA induction in MyePro cells, both of which are attributed to the endonuclease activity of ANG. 6 Therefore, the cleavage of ANG on rRNA or tRNA could be a very basic mechanism of action for this multifunctional protein. This study extends the substrates of this protein to include miRNA, which suggests the presence of a novel mode of miRNA turnover in mammalian cells that might be important for rapid changes in miRNA expression profiles during numerous physiological or pathological processes. Although currently we could not identify more different kinds of miRNAs as ANG's substrate, further study would be of interest to explore whether ANG has additional non-coding RNA targets, including long and circular RNAs.
As an endonuclease, the cleavage site of ANG in miR-141 must be identified. According to our results from the cell-free cleavage assay, ANG preferably cuts miR-141 at the A 5 /C 6 , U 7 /G 8 , and U 14 /A 15 sites.
However, the precise digestion site in the cellular content remains unknown. We attempted to assess the digestion products of miR-141 by introducing 5 0 Cy5-or 3 0 Cy5-labeled miR-141 into cells, but the cleavage pattern of Cy5-labeled miR-141 differs from that observed in the cell-free system (data not shown). Such a discrepancy could be due to the interference of Cy5 because the cyanine-dye-labeled oligonucleotide probe could accumulate at the surface of mitochondria; 37 alternatively, this modification might change the miR-141 structure in cells as the results showed that miR-141 in a structured  buffer may create a secondary structure that contains a duplex to block ANG digestion ( Figure S6). Nevertheless, ANG's recognition and cleavage of its substrate may not be restricted to a specific sequence motif. For example, ANG cleaves 47S pre-rRNA at the A 0 site of "CUC/UUC," 11 but cleaves tRNA at the CA motif of conserved single-stranded 3 0 CCA termini and the anticodon loop. 13,38 By contrast, in the cell-free system, ANG degrades 28S and 18S rRNAs into a pattern ranging from 100 to 500 nt in length. 39 These findings suggest that auxiliary factors may assist in its cleavage or that a spe-cific spatial structure may be formed in cells that permit exposure of its digestion site. In addition, a technical limitation is the current lack of an efficient method to effectively capture and analyze RNAs smaller than 18 nt. 40,41 In fact, we do not know how miRNases, such as exoribonuclease SDN1 30 or endonuclease TSN, 36  not affect pri-and pre-miR-141 in cells, digestion is likely to occur during the single-strand formation stage ( Figures 2B and 2C); moreover, ANG does not affect the double-stranded versions ( Figures 3C  and 3D). It is speculated that ANG cleavage may not occur after miR-ISC formation because we observed profound effects on miR-141 target expression. 25 Therefore, there may be a competing mechanism between ANG and certain RISC components for binding miR-141 at the time of RISC assembly. It is also interesting to observe that ANG only targets miR-141 specifically, although the sequences of miR-200 family members are quite similar. One possible explanation is that these miRNAs may incorporate into different RNA-protein or miRNA-RNA complexes, and the cleavage sites of other members might be shielded from ANG digestion. While, methylation or other types of modification may also affect the interaction between ANG and the members of the miR-200 family, at least methylated nuclei acids were resisted to ANG cleavage as shown in Figure 3E.
Angiogenesis and cancer cell proliferation are required for tumor development, and ANG serves as a critical regulator both in angiogenesis and tumorigenesis. Ribonuclease activity, basement membrane degradation, signaling transduction, and nuclear translocation are considered to be four aspects for the process of ANG-induced angiogenesis. 42 The secreted ANG undergoes binding to the cell surface actin to promote basement membrane and extracellular matrix degradation, and receptor Plexin-B2 43 mediated endocytosis and appropriate subcellular localization to activate cell-type-specific signaling and RNA processing. Besides signaling transduction activation, RNA processing is the most critical activity for ANG to participate in the cellular responses involved in angiogenesis and tumor development, including rRNA transcription and tiRNA production, as we described in the Introduction. When the activity of ribonuclease or nuclear translocation was deprived, ANG is unable to induce angiogenesis 9 or tumorigenesis. 44,45 Meanwhile, ANG can also cleave 47S pre-rRNA 11 and Figure 7. A schematic of ANG-mediated endonucleolytic decay of miR-141 promotes angiogenesis and tumor growth This schematic shows that ANG cleaves mature miR-141 via direct binding and endonucleolytic digestion depending on its ribonuclease activity. By regulating miR-141 level and its angiogenesis-related targets GAB1, GATA6, TGF-b, and NRP1, ANG promotes the processes of angiogenesis thereby promoting tumor progression.
degrade 28S and 18S rRNAs, 39 bind or process mRNA, 46 and cleave an unknown group of RNAs. 47 Here, we report that miRNA as a new substrate of ANG, which is a complement for the mechanism of ANG on nucleic acid metabolism. Furthermore, the function of miR-141 was identified to inhibit angiogenesis 25 and tumor progression, 48 demonstrating the rational role of ANG-/miR-141 axis in CRC progression.
In conclusion, our study provides new insights into the functions and mechanisms of ANG and miR-141 in angiogenesis. We not only identified ANG as an endonuclease involved in miRNA turnover, but we also elucidated a novel angiogenesis regulatory axis of ANG, miR-141, and targets in CRC (Figure 7). Based on these findings, we believe that ANG and miR-141 may have diagnostic and therapeutic values in CRC.

Cell culture and treatment
The HUVECs were cultured as described previously. 49 The nuclear translocation of ANG in endothelial cells is required for ANGinduced angiogenesis and is strictly dependent on the cell density.
To identify miRNAs that responded to ANG stimulation, the HUVECs were cultured at a density of 5,000 cells/cm 2 and were starved with FBS-free medium overnight. Subsequently, the cells were incubated with 1 mg/mL of ANG for 6 h and then harvested for miRNA screening. For miRNA detection assays, 1 mg/mL of recombinant human ANG, ribonuclease mutated variant ANG-K40I or ANG-H114A, RNase-4 or RNase A, and 200 mM neomycin or 4 U/mL RI were used to treat HUVECs for 24 h after overnight starvation using FBS-free medium. The recombinant human ANG and its mutants ANG-K40I or ANG-H114A were produced with the bacterial expression system as described previously and confirmed by mass spectrometry nanlysis. 3

miRNA screening
The treated cells were harvested with trypsin and washed with PBS. They were then resuspended in PBS at a density of 2 Â 10 7 /mL. Ten microliters of the cell suspension was lysed in 90 mL of lysis buffer (15 mM Tris, 10 mM NaCl, 3 mM MgCl 2 , 0.3% NP-40) for 10 min on ice, heated at 99 C for 10 min, and then placed on ice again after heating. Six microliters of lysate was used as a reverse transcription www.moleculartherapy.org template. 50 After undergoing reverse transcription with the Human MegaPlex reverse transcription (RT) Primer pools (Applied Biosystems), the TaqMan Low-Density Array Human MicroRNA Cards Set v.2.0 (Applied Biosystems, cat. no. 4400238), which contains 667 mature miRNA probes and four control probes, was used to perform the miRNA expression analysis on the products.

miRNA detection
The treated cells were harvested, and total RNAs were isolated with TRIzol reagent (Life Technologies). The expression levels of individual mature and primary miRNAs were determined by qRT-PCR using the TaqMan miRNA assay kit (Life Technologies). The pre-miRNA level was determined by qRT-PCR using the miScript Precursor Assays kit (QIAGEN). U6 small nuclear RNA was used as an internal control. The data were analyzed using the DDCt method.

RIP for miRNA
RIP was performed according to the instructions provided with the Magna RIP kit (Millipore). In brief, after being washed with PBS and crosslinked with 1% formaldehyde, ANG-overexpressing SW480 cells were lysed in RIP lysis buffer. The cell lysate supernatant was incubated with protein A/G magnetic beads bound to rabbit anti-ANG polyclonal antibodies (homemade) 7 or normal IgG. The protein-RNA-bead complexes were collected for RNA detection via qRT-PCR or protein detection via western blotting.

miRNA northern blotting
Northern blotting was performed as described previously 51 with modifications. In brief, 30 mg total RNA samples resolved on a 17% urea polyacrylamide denaturing gel were transferred to a positively charged nylon membrane (Roche), UV-crosslinked, and probed with DIG-labeled locked nucleic acid (LNA)-modified oligonucleotide probes (Exiqon) overnight at 50 C. The membrane was washed twice at room temperature with low stringency buffer (2 Â SSC, 0.1% SDS) for 5 min, washed twice at 65 C-68 C with high stringency buffer (0.5 Â SSC, 0.1% SDS) for 15 min, and washed once at room temperature with washing buffer for 5 min. After blocking for more than 30 min with blocking solution and incubation with DIG antibodies (Roche), the membrane was washed once with washing buffer for 15 min and balanced in detection buffer (Roche) for 5 min, followed by incubation with CDP-Star substrate (Roche). The fluorescent signal on the membrane was recorded using the G:BOX Chemi XRQ gel doc system (Syngene).
Cell-free cleavage assay RNA oligos or duplexes were synthesized by Biomics Biotechnologies and GenePharma. 5 0 Cy5-or 3 0 Cy5-labeled RNA oligos were synthesized by Integrated Device Technology. DNA oligos were synthesized by Sangon Biotech. Typically, in a 4-mL cleavage system, the substrate (2.5 ng/mL of Cy5-labeled RNA or 25 ng/mL of unlabeled RNA) was incubated with ANG protein (10 ng/mL) in DEPC-treated H 2 O at 37 C for 30 min or for the indicated time. The reactions were then terminated by the addition of 1 volume of urea loading buffer and put on ice for 5 min; the solution was then subjected to denaturing urea polyacrylamide gel electrophoresis (7 M urea-denaturing 15%-20% polyacrylamide gel with 0.5 Â TBE buffer). Unlabeled oligos in the gels were stained with SYBR Gold dye (Life Technologies), and Cy5labeled oligos in the gels were scanned using a fluorescence scanner (LI-COR Biosciences). The alkaline hydrolysis ladder was generated by incubating the labeled substrate in alkaline hydrolysis buffer (50 mM NaOH in urea loading buffer) at 80 C for 2-15 min.

EMSA
An EMSA was employed to analyze RNA-protein interactions in vitro. Cy5-labeled miR-141 (5 ng/mL) was incubated with ANG (10 ng/mL) or ANG-K40I (10 ng/mL) protein at 37 C for 5-30 min, and Cy5-miR-141 incubated without the protein was used as a control. The specificity of the interaction was tested via blocking studies in the presence of RI or competition studies in the presence of an excess amount of unlabeled miR-141. The following concentrations of RI were used: 0.56 U/mL (1Â), 1.12 U/mL (2Â), and 2.24 U/mL (4Â). Furthermore, 20 ng (1Â), 40 ng (2Â), or 80 ng (4Â) of unlabeled miR-141 were added to compete with the 20 ng of Cy5-miR-141 in the competition reactions. The blocking studies and competition studies were performed during 30 min of incubation at 37 C. The reactions were put on ice for 5 min and then underwent native polyacrylamide gel electrophoresis. RNA-protein complexes and free RNA were visualized using Bio-Rad ChemiDoc MP.

In vitro angiogenesis assay
In vitro angiogenesis assays that assessed the processes of vessel endothelial cell proliferation, migration, and tube formation were performed as described previously. 25 The cell proliferation activity of HUVECs was determined using the Cell Counting Kit-8 (Dojindo). Cell migration activity was assessed using the real-time cell migration assays with the xCELLigence RTCA DP instrument (Roche) in CIMplate 16. The tube formation assay was assessed by the capillary-like structure formation ability of HUVECs on Growth Factor Reduced Matrigel (BD Biosciences).

In vivo angiogenesis assay
The in vivo angiogenesis assay was performed using a Matrigel plug assay as described previously. 25 ANG alone (1 mg/mL) or ANG containing 100 mM agomir-141 (GenePharma) was resuspended in 30 mL of PBS and mixed with 500 mL of Matrigel Basement Membrane Matrix (BD Biosciences) containing 15 units of heparin (Sigma Aldrich). The Matrigel mixture was injected subcutaneously into C57 BL/6 mice (5-7 weeks old) along the abdominal midline. After 6-8 days, the animals were sacrificed to harvest the plug. The angiogenesis ability was quantified by analyzing the immunohistochemical staining of blood vessels with anti-human CD31 antibodies (Abcam). The mice were manipulated and cared for according to the guidelines and protocols approved by the Medical Experimental Animal Care Commission of Zhejiang University (Approval ID ZJU201402-1-01-033).

Tissue specimens
The fresh samples of CRC and the adjacent normal tissues were provided by the tissue bank of the Second Affiliated Hospital of Zhejiang University School of Medicine. Formalin-fixed paraffin-embedded CRC tissues were provided by the tissue bank of the First Affiliated Hospital of Zhejiang University School of Medicine. Plasma samples from CRC patients and healthy individuals were obtained from the Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. All studies were approved by the Ethics Committee of the Zhejiang University School of Medicine (no. 2018-020).

Statistical analysis
The data analyses were performed using GraphPad Prism 8 software.
The data are presented as the means ± SD or percentages of the control ± SD from at least three independent experiments. The differences in the cell migration curve were analyzed with a two-way ANOVA. The differences in miRNA or mRNA expression among more than two groups and cell proliferation at different time points were analyzed with a one-way ANOVA. The differences between two groups were determined using the two-tailed unpaired Student's t test. For the mouse experiments, n denotes the number of mice, and p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01).